Although cellular respiration is set by metabolic need during normoxia, evidence suggests that cells can adapt to hypoxia by reducing their energy demand, thereby lowering the use of ATP and the need for O2. At reoxygenation, normal metabolic processes are restored and cellular activity recovers. An ability to reduce energy demands in hypoxia while preserving high energy phosphate levels (hypoxic adaptation) may be protective during severe hypoxia by conserving ATP for essential processes. Hypoxic adaptation requires a cellular O2 sensor capable of detecting PO2. Although multiple sensors likely exist, data suggest that cytochrome oxidase acts as the sensor during hypoxic adaptation. Specific Aim 1 will test whether cytochrome oxidase functions as the O2 sensor during hypoxia by decreasing its apparent Vmax. This will be tested using inhibitors that reduce the Vmax of the oxidase during normoxia, determining whether these activate the hypoxic adaptation response. Activation of the O2 sensor during hypoxia must be coupled to subsequent activation of an intracellular signaling cascade, which ultimately inhibits ATP utilization. Specific Aim 2 will test the hypothesis that reactive oxygen species (ROS) function as a second messenger in this signaling pathway. The PO2-dependent ROS generation in intact cells will be studied and correlated with the function of the oxidase. Other studies will confirm whether mitochondria are the source of the ROS and will link these signals to the activation of the hypoxic adaptation response. Studies with isolated mitochondria will identify the sites and mechanisms of PO2-dependent ROS generation during hypoxia. Collectively, these studies will clarify the mechanisms of mitochondrial ROS generation during hypoxia and link these signals to the function of cytochrome oxidase and the hypoxic adaptation response. The hypothesis is that signaling elements downstream of ROS lead to inhibition of ATP-dependent enzyme systems. Specific Aim 3 will begin to test the hypothesis that protein kinases function as downstream signaling elements in the hypoxic response. The involvement of protein kinase C in this pathway will be tested, based on previous studies demonstrating its activation by ROS or by hypoxia. The long term goal of this project is to identify O2 sensing mechanisms and the downstream signaling sequence involved in hypoxic adaptation. These studies will identify a novel pathway of cellular O2 detection, and may help clarify understanding of how cells adapt to lowered O2 conditions.